Assays for detecting analytes in samples and kits and compositions related thereto

ABSTRACT

The present invention provides methods of detecting analytes using particles having different physico-chemical properties, such as buoyancy, size, density, spectral characteristics, and/or binding properties, in solution-based sandwich assays and solution-based competition assays. The methods can be performed using rotors and bench-top centrifuges and provide for rapid, qualitative and quantitative detection of analytes. The present invention also provides kits that can be used to perform the methods, and mixtures containing particles suitable for the methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/158,901, filed May 19, 2016, which is a continuation of U.S. patentapplication Ser. No. 12/971,968, filed on Dec. 17, 2010 (now abandoned),which claims the benefit of priority of U.S. Provisional PatentApplication No. 61/287,637, filed Dec. 17, 2009, each of which areherein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

In many fields of endeavor, and notably biomedical sciences, veterinarysciences, and environmental sciences, it is important to be able todetect molecules of interest (i.e., analytes) in samples that have beencollected, for example, from test subjects (e.g., patients, laboratoryand farm animals, pets, etc.) or the environment. To meet these needs,many different assays have been developed, ranging from lateral flowdevices (e.g., home pregnancy tests), to immunoprecipitations andELISAs, to mass spectrometry. These assays have proven very useful, butoften suffer from a number of important drawbacks, such as being costlyto implement, time consuming, technically complicated, hard to scale upfor large sample sizes or large numbers of samples, etc.

There remains a need in the art for new assays for detecting analytes insamples.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that particleshaving different physico-chemical properties, such as buoyancy, size,density, spectral characteristics, and/or binding properties, can beused in solution-based sandwich assays for rapid, qualitative andquantitative detection of analytes. The present invention is also based,in part, on the discovery that particles having differentphysico-chemical properties, such as buoyancy, size, density, spectralcharacteristics, and/or binding properties, can be used insolution-based competition assays for rapid, qualitative andquantitative detection of analytes. Accordingly, the present inventionprovides methods of detecting analytes, kits that can be used to performsuch methods, and mixtures containing particles suitable for suchmethods.

In one aspect, the invention provides methods of detecting an analyte.In certain embodiments, the methods (e.g., solution-based sandwichassays) comprise:

mixing a sample with a population of first particles and a population ofsecond particles to form a suspension, wherein the particles are capableof forming multi-particle complexes comprising a first particle, asecond particle, and an analyte,

removing multi-particle complexes formed upon said mixing from saidsuspension, and

detecting the presence of first and/or second particles remaining insuspension,

wherein a decrease in amount of first and/or second particles insuspension is indicative of the presence of the analyte in the sample.

In certain embodiments, the first and/or second particles are detectablein suspension. For example, in certain embodiments, the first and/orsecond particles scatter light or comprise a detectable color (e.g.,visually or spectroscopically detectable color). In certain embodiments,the first and/or second particles comprise a label (e.g., fluorescentlabel). In certain embodiments, both the first and second particlescomprise a label (e.g., different fluorescent labels or differentchromophores). In certain embodiments, the first particles comprise adonor chromophore and the second particles comprise an acceptorchromophore, or vice verse, wherein the donor and acceptor chromophoresare suitable for detecting interaction between the first and secondparticles by means of Förster Resonance Energy Transfer (FRET) analysis.

In certain embodiments, the first and/or second particles are colloidalparticles (e.g., colloidal nanoparticles, nanotubes, core-shellstructure particles, or hollow nanospheres). In certain embodiments, thefirst and/or second particles comprise gold, silver, platinum, copper,or mixed metal. In certain embodiments, the first and/or secondparticles comprise latex, polystyrene, polycarbonate, polyvinylidenefluoride, silica, a polymer having properties similar to any of theforegoing polymers, or a composite thereof. In certain embodiments, thefirst particles comprise gold, silver, platinum, copper, or mixed metal,and the second particles comprise latex, polystyrene, polycarbonate,polyvinylidene fluoride, silica, a polymer having properties similar toany of the foregoing polymers, or a composite thereof.

In certain embodiments, the first particles and the second particleshave different physico-chemical properties. For example, in certainembodiments, the first particles have a greater buoyancy than the secondparticles, or vice versa. In certain embodiments, the first particlesare smaller than the second particles, or vice versa. For example, incertain embodiments, the second particles have a diameter about 5 toabout 50 times larger than the diameter of the first particles.Alternatively, in certain embodiments, the first particles have adiameter about 5 to about 50 times larger than the diameter of thesecond particles. In certain embodiments, the first particles have agreater density than the second particles, or vice versa. In certainembodiments, the first particles are smaller and more buoyant than thesecond particles. In certain embodiments, the first particles aresmaller, more dense, and more buoyant than the second particles.

In certain embodiments, the first particles, second particles, or bothfirst and second particles comprise an analyte-binding agent, such as anantibody, antigen, polypeptide, polynucleotide, nucleoprotein, aptamer,or ligand (e.g., a carbohydrate, lipid, steroid, vitamin, or other smallmolecule ligand). In certain embodiments, the first and second particlescomprise different analyte-binding agents. In certain embodiments, thefirst and second particles comprise analyte-binding agents capable ofbinding to different parts of the same analyte (e.g., different domains,different epitopes, different subunits or molecules in a complexanalyte, etc.). In certain embodiments, the first and second particlescomprise analyte-binding agents capable of simultaneously binding to thesame analyte. In certain embodiments, the first particles comprise afirst antibody capable of recognizing a first epitope on an analyte, thesecond particles comprise a second antibody capable of recognizing asecond epitope on the same analyte, and the first and second epitopesare different (e.g., non-overlapping). For example, the first and secondepitopes can be on different surfaces of a simple analyte or differentsubunits or molecules in a complex analyte. In other embodiments, thefirst particles comprise an antigen capable of being recognized by ananalyte (e.g., an antibody analyte, such as a disease-specific antibodyor an auto-antibody) and the second particles comprise a protein orantibody capable of recognizing the analyte. For example, the secondparticle can comprise a protein that is an antibody-binding protein,such as Protein A, Protein G, or Protein L, or an antibody that binds toan antibody constant region (e.g., an anti-IgG or anti-IgM antibody).

In certain embodiments, removing said multi-particle complexes comprisesallowing gravity to pellet said complexes. In other embodiments,removing said multi-particle complexes comprises applying a force. Forexample, in certain embodiments, removing said multi-particle complexescomprises applying centrifugal force to said suspension (e.g., byspinning the suspension in a rotor). In certain embodiments, the gravityor force applied pellets said multi-particle particle complexes but doesnot pellet first particles and second particles that are not present inone of said multi-particle complexes. In certain embodiments, thegravity or force applied pellets said multi-particle complexes andeither said first particles (e.g., free first particles and firstparticles present in a multi-particle complex) or said second particles(e.g., free second particles and second particles present in amulti-particle complex), but not both said first particles and saidsecond particles.

In certain embodiments, the analyte is present in a biological sample(e.g., blood, serum, urine, etc.) or an environmental sample (e.g., asample of ground water, river, lake, waste water, etc.). In certainembodiments, the analyte is a marker (e.g., an antigen marker orantibody marker) for a disease. For example, in certain embodiments, theanalyte is a cancer-related antigen, a viral antigen, a bacterialantigen, a fungal antigen, an autoimmune-associated antigen, acardiovascular disease-associated antigen, or an antibody to any of theforegoing antigens.

In certain embodiments, the invention provides methods (e.g.,solution-based sandwich assays) for detecting an analyte, the methodscomprising:

mixing a sample with a population of first particles and a population ofsecond particles to form a suspension, wherein the particles are capableof forming multi-particle complexes comprising a first particle, asecond particle, and an analyte,

removing said second particles from said suspension, and

detecting the presence of first particles remaining in suspension,

wherein a decrease in amount of first particles in suspension isindicative of the presence of the analyte in the sample.

In certain embodiments, the first particles are detectable insuspension. For example, in certain embodiments, the first particlesscatter light or comprise a detectable color (e.g., visually orspectroscopically detectable color). In certain embodiments, the firstparticles comprise a label (e.g., a fluorescent label). In certainembodiments, both the first and second particles comprise a label (e.g.,different fluorescent labels or different chromophores). In certainembodiments, the first particles comprise a donor chromophore and thesecond particles comprise an acceptor chromophore, or vice verse,wherein the donor and acceptor chromophores are suitable for detectinginteraction between the first and second particles by means of FRETanalysis.

In certain embodiments, the first particles are colloidal particles(e.g., colloidal nanoparticles, nanotubes, core-shell structureparticles, or hollow nanospheres). In certain embodiments, the first andsecond particles are colloidal particles (e.g., colloidal nanoparticles,nanotubes, core-shell structure particles, or hollow nanospheres). Incertain embodiments, the first particles comprise gold, silver,platinum, copper, or mixed metal. In certain embodiments, the firstparticles comprise latex, polystyrene, polycarbonate, polyvinylidenefluoride, silica, a polymer having properties similar to any of theforegoing polymers, or a composite thereof. In certain embodiments, thefirst and second particles comprise latex, polystyrene, polycarbonate,polyvinylidene fluoride, silica, a polymer having properties similar toany of the foregoing polymers, or a composite thereof. In certainembodiments, the first particles comprise gold, silver, platinum,copper, or mixed metal, and the second particles comprise latex,polystyrene, polycarbonate, polyvinylidene fluoride, silica, a polymerhaving properties similar to any of the foregoing polymers, or acomposite thereof.

In certain embodiments, the first particles and second particles havedifferent physico-chemical properties. For example, in certainembodiments, the first particles have a greater buoyancy than the secondparticles. In certain embodiments, the first particles are smaller thanthe second particles. For example, in certain embodiments, the secondparticles have a diameter about 5 to about 50 times larger than thediameter of the first particles. In certain embodiments, the firstparticles have a greater density than the second particles. In otherembodiments, the second particles have a greater density than the firstparticles. In certain embodiments, the first particles are smaller andmore buoyant than the second particles. In certain embodiments, thefirst particles are smaller, more dense, and more buoyant than thesecond particles.

In certain embodiments, the first particles, second particles, or boththe first and second particles comprise an analyte-binding agent, suchas an antibody, antigen, polypeptide, polynucleotide, nucleoprotein,aptamer, or ligand (e.g., a carbohydrate, lipid, steroid, vitamin, orother small molecule ligand). In certain embodiments, the first andsecond particles comprise different analyte-binding agents. In certainembodiments, the first and second particles comprise analyte-bindingagents capable of binding to different parts of the same analyte (e.g.,different domains, different epitopes, different subunits or moleculesin a complex analyte, etc.). In certain embodiments, the first andsecond particles comprise analyte-binding agents capable ofsimultaneously binding to the same analyte. In certain embodiments, thefirst particles comprise a first antibody capable of recognizing a firstepitope on an analyte, the second particles comprise a second antibodycapable of recognizing a second epitope on the same analyte, and thefirst and second epitopes are different (e.g., non-overlapping). Forexample, the first and second epitopes can be on different surfaces of asimple analyte or different subunits or molecules in a complex analyte.In other embodiments, the first particles comprise an antigen capable ofbeing recognized by an analyte (e.g., an antibody analyte, such as adisease-specific antibody or an auto-antibody) and the second particlescomprise a protein or antibody capable of recognizing the analyte. Forexample, the second particle can comprise a protein that is anantibody-binding protein, such as Protein A, Protein G, or Protein L, oran antibody that binds to an antibody constant region (e.g., an anti-IgGor anti-IgM antibody).

In certain embodiments, removing said second particles comprisesremoving said multi-particle complexes. In certain embodiments, removingsaid second particles comprises allowing gravity to pellet said secondparticles (and said multi-particle complexes). In other embodiments,removing said second particles comprises applying a force. For example,in certain embodiments, removing said second particles comprisesapplying centrifugal force to said suspension (e.g., by spinning thesuspension in a rotor). In certain embodiments, the gravity or forceapplied pellets said second particles but does not pellet firstparticles that are not complexed to one of the second particles.

In certain embodiments, the analyte is present in a biological sample(e.g., blood, serum, urine, etc.) or an environmental sample (e.g., asample of ground water, river, lake, waste water, etc.). In certainembodiments, the analyte is a marker (e.g., an antigen marker orantibody marker) for a disease. For example, in certain embodiments, theanalyte is a cancer-related antigen, a viral antigen, a bacterialantigen, a fungal antigen, an autoimmune-associated antigen, acardiovascular disease-associated antigen, or an antibody to any of theforegoing antigens.

In other embodiments, the invention provides methods (e.g.,solution-based competition assays) for detecting an analyte, the methodscomprising:

mixing a sample with a population of first particles and a population ofsecond particles to form a suspension, wherein the first particlescomprise an analyte, and wherein the particles are capable of formingmulti-particle complexes comprising a first particle and a secondparticle,

removing multi-particle complexes formed upon said mixing from saidsuspension, and

detecting the presence of first and/or second particles remaining insuspension,

wherein an increase in amount of first and/or second particles insuspension is indicative of the presence of the analyte in the sample.

In certain embodiments, the first and/or second particles are detectablein suspension. For example, in certain embodiments, the first and/orsecond particles scatter light or comprise a detectable color (e.g.,visually or spectroscopically detectable color). In certain embodiments,the first and/or second particles comprise a label (e.g., fluorescentlabel). In certain embodiments, both the first and second particlescomprise a label (e.g., different fluorescent labels or differentchromophores). In certain embodiments, the first particles comprise adonor chromophore and the second particles comprise an acceptorchromophore, or vice verse, wherein the donor and acceptor chromophoresare suitable for detecting interaction between the first and secondparticles by means of FRET analysis.

In certain embodiments, the first and/or second particles are colloidalparticles (e.g., colloidal nanoparticles, nanotubes, core-shellstructure particles, or hollow nanospheres). In certain embodiments, thefirst and/or second particles comprise gold, silver, platinum, copper,or mixed metal. In certain embodiments, the first and/or secondparticles comprise latex, polystyrene, polycarbonate, polyvinylidenefluoride, silica, a polymer having properties similar to any of theforegoing polymers, or a composite thereof. In certain embodiments, thefirst particles comprise gold, silver, platinum, copper, or mixed metal,and the second particles comprise latex, polystyrene, polycarbonate,polyvinylidene fluoride, silica, a polymer having properties similar toany of the foregoing polymers, or a composite thereof.

In certain embodiments, the first particles and the second particleshave different physico-chemical properties. For example, in certainembodiments, the first particles have a greater buoyancy than the secondparticles, or vice versa. In certain embodiments, the first particlesare smaller than the second particles, or vice versa. For example, incertain embodiments, the second particles have a diameter about 5 toabout 50 times larger than the diameter of the first particles.Alternatively, in certain embodiments, the first particles have adiameter about 5 to about 50 times larger than the diameter of thesecond particles. In certain embodiments, the first particles have agreater density than the second particles, or vice versa. In certainembodiments, the first particles are smaller and more buoyant than thesecond particles. In certain embodiments, the first particles aresmaller, more dense, and more buoyant than the second particles.

In certain embodiments, the second particles comprise an analyte-bindingagent, such as an antibody, antigen, polypeptide, polynucleotide,nucleoprotein, aptamer, or ligand (e.g., a carbohydrate, lipid, steroid,vitamin, or other small molecule ligand). In certain embodiments, theanalyte-binding agent is capable of binding to analyte present on saidfirst particles.

In certain embodiments, removing said multi-particle complexes comprisesallowing gravity to pellet said multi-particle complexes. In otherembodiments, removing said multi-particle complexes comprises applying aforce. For example, in certain embodiments, removing said multi-particlecomplexes comprises applying centrifugal force to said suspension (e.g.,by spinning the suspension in a rotor). In certain embodiments, thegravity or force applied pellets said multi-particle complexes but doesnot pellet first particles and second particles that are not present inone of said multi-particle complexes. In certain embodiments, thegravity or force applied pellets said multi-particle complexes andeither said first particles (e.g., free first particles and firstparticles present in a multi-particle complex) or said second particles(e.g., free second particles and second particles present in amulti-particle complex), but not both of said first particles and saidsecond particles.

In certain embodiments, the analyte is present in a biological sample(e.g., blood, serum, urine, etc.) or an environmental sample (e.g., asample of ground water, river, lake, waste water, etc.). In certainembodiments, the analyte is a marker (e.g., an antigen marker or anantibody marker) for a disease. For example, in certain embodiments, theanalyte is a cancer-related antigen, a viral antigen, a bacterialantigen, a fungal antigen, an autoimmune-associated antigen, acardiovascular disease-associated antigen, or an antibody to any of theforegoing antigens.

In still other embodiments, the invention provides methods (e.g.,solution-based competition assays) for detecting an analyte, the methodscomprising:

mixing a sample with a population of first particles and a population ofsecond particles to form a suspension, wherein the first particlescomprise an analyte, and wherein the particles are capable of formingmulti-particle complexes comprising a first particle and a secondparticle,

removing said second particles from said suspension, and

detecting the presence of first particles remaining in suspension,

wherein an increase in amount of first particles in suspension isindicative of the presence of the analyte in the sample.

In certain embodiments, the first particles are detectable insuspension. For example, in certain embodiments, the first particlesscatter light or comprise a detectable color (e.g., visually orspectroscopically detectable color). In certain embodiments, the firstparticles comprise a label (e.g., a fluorescent label). In certainembodiments, both the first and second particles comprise a label (e.g.,different fluorescent labels or different chromophores). In certainembodiments, the first particles comprise a donor chromophore and thesecond particles comprise an acceptor chromophore, or vice verse,wherein the donor and acceptor chromophores are suitable for detectinginteraction between the first and second particles by means of FRETanalysis.

In certain embodiments, the first and/or second particles are colloidalparticles (e.g., colloidal nanoparticles, nanotubes, core-shellstructure particles, or hollow nanospheres). In certain embodiments, thefirst and/or second particles comprise gold, silver, platinum, copper,or mixed metal. In certain embodiments, the first and/or secondparticles comprise latex, polystyrene, polycarbonate, polyvinylidenefluoride, silica, a polymer having properties similar to any of theforegoing polymers, or a composite thereof. In certain embodiments, thefirst particles comprise gold, silver, platinum, copper, or mixed metal,and the second particles comprise latex, polystyrene, polycarbonate,polyvinylidene fluoride, silica, a polymer having properties similar toany of the foregoing polymers, or a composite thereof.

In certain embodiments, the first particles and the second particleshave different physico-chemical properties. For example, in certainembodiments, the first particles have a greater buoyancy than the secondparticles. In certain embodiments, the first particles are smaller thanthe second particles. For example, in certain embodiments, the secondparticles have a diameter about 5 to about 50 times larger than thediameter of the first particles. In certain embodiments, the firstparticles have a greater density than the second particles. In otherembodiments, the second particles have a greater density than the firstparticles. In certain embodiments, the first particles are smaller andmore buoyant than the second particles. In certain embodiments, thefirst particles are smaller, more dense, and more buoyant than thesecond particles.

In certain embodiments, the second particles comprise an analyte-bindingagent, such as an antibody, antigen, polypeptide, polynucleotide,nucleoprotein, aptamer, or ligand (e.g., a carbohydrate, lipid, steroid,vitamin, or other small molecule ligand). In certain embodiments, theanalyte-binding agent is capable of binding to analyte present on saidfirst particles.

In certain embodiments, removing said second particles comprisesremoving said multi-particle complexes. In certain embodiments, removingsaid second particles comprises allowing gravity to pellet said secondparticles (and said multi-particle complexes). In other embodiments,removing said second particles comprises applying a force. For example,in certain embodiments, removing said second particles comprisesapplying centrifugal force to said suspension (e.g., by spinning thesuspension in a rotor). In certain embodiments, the gravity or forceapplied pellets said second particles but does not pellet firstparticles that are not complexed to one of the second particles.

In certain embodiments, the analyte is present in a biological sample(e.g., blood, serum, urine, etc.) or an environmental sample (e.g., asample of ground water, river, lake, waste water, etc.). In certainembodiments, the analyte is a marker (e.g., an antigen marker or anantibody marker) for a disease. For example, in certain embodiments, theanalyte is a cancer-related antigen, a viral antigen, a bacterialantigen, a fungal antigen, an autoimmune-associated antigen, acardiovascular disease-associated antigen, or an antibody to any of theforegoing antigens.

In another aspect, the invention provides kits comprising a populationof first particles and a population of second particles suitable for usein methods of the invention. In certain embodiments, the first andsecond particles are capable of forming multi-particle complexes. Incertain embodiments, the first and second particles are suitable for usein solution-based competition assays. For example, in certainembodiments, the first and second particles are capable of formingmulti-particle complexes wherein free analyte disrupts (e.g.,competitively inhibits) formation of said multi-particle complexes. Inother embodiments, the first and second particles are suitable for usein solution-based sandwich assays (e.g., direct or indirect sandwichassays). For example, in certain embodiments, the first and secondparticles are capable of forming multi-particle complexes comprising afirst particle, a second particle, and an analyte. In certain relatedembodiments, the first and second particles are capable of formingmulti-particle complexes, wherein said first particle and said secondparticle each bind to the same analyte, and wherein said analyte linkssaid first particle to said second particle.

In certain embodiments, said first and/or second particles aredetectable in suspension. For example, in certain embodiments, the firstand/or second particles scatter light or comprise a detectable color(e.g., visually or spectroscopically detectable color). In certainembodiments, the first and/or second particles comprise a label (e.g., afluorescent label). In certain embodiments, both the first and secondparticles comprise a label (e.g., different fluorescent labels ordifferent chromophores). In certain embodiments, the first particlescomprise a donor chromophore and the second particles comprise anacceptor chromophore, or vice verse, wherein the donor and acceptorchromophores are suitable for detecting interaction between the firstand second particles by means of Förster Resonance Energy Transfer(FRET) analysis.

In certain embodiments, the first and/or second particles are colloidalparticles (e.g., colloidal nanoparticles, nanotubes, core-shellstructure particles, or hollow nanospheres). In certain embodiments, thefirst and/or second particles comprise gold, silver, platinum, copper,or mixed metal. In certain embodiments, the first and/or secondparticles comprise latex, polystyrene, polycarbonate, polyvinylidenefluoride, silica, a polymer having properties similar to any of theforegoing polymers, or a composite thereof. In certain embodiments, thefirst particles comprise gold, silver, platinum, copper, or mixed metal,and the second particles comprise latex, polystyrene, polycarbonate,polyvinylidene fluoride, silica, a polymer having properties similar toany of the foregoing polymers, or a composite thereof.

In certain embodiments, the first particles and the second particleshave different physico-chemical properties. For example, in certainembodiments, the first particles have a greater buoyancy than the secondparticles. In certain embodiments, the first particles are smaller thanthe second particles. For example, in certain embodiments, the secondparticles have a diameter about 5 to about 50 times larger than thediameter of the first particles. In other embodiments, the firstparticles have a diameter about 5 to about 50 times larger than thediameter of the second particles. In certain embodiments, the firstparticles have a greater density than the second particles, or viceversa. In certain embodiments, the first particles are smaller and morebuoyant than the second particles. In certain embodiments, the firstparticles are smaller, more dense, and more buoyant than the secondparticles.

In certain embodiments, the first particles comprise an analyte and thesecond particles comprise a corresponding analyte-binding agent, such asan antibody, antigen, polypeptide, polynucleotide, nucleoprotein,aptamer, or ligand (e.g., a carbohydrate, lipid, steroid, vitamin, orother small molecule ligand). In certain embodiments, both the first andsecond particles comprise an analyte-binding agent, such as an antibody,antigen, polypeptide, polynucleotide, nucleoprotein, aptamer, or ligand(e.g., a carbohydrate, lipid, steroid, vitamin, or other small moleculeligand), wherein the analyte-binding agents bind to the same analyte.

In certain embodiments, the first and second particles comprisedifferent analyte-binding agents. In certain embodiments, the first andsecond particles comprise analyte-binding agents capable of binding todifferent parts of the same analyte (e.g., different domains, differentepitopes, different subunits or molecules in a complex analyte, etc.).In certain embodiments, the first and second particles compriseanalyte-binding agents capable of simultaneously binding to the sameanalyte. In certain embodiments, the first particles comprise a firstantibody capable of recognizing a first epitope on an analyte, thesecond particles comprise a second antibody capable of recognizing asecond epitope on the same analyte, and the first and second epitopesare different (e.g., non-overlapping). For example, the first and secondepitopes can be on different surfaces of a simple analyte or differentsubunits or molecules in a complex analyte. In other embodiments, thefirst particles comprise an antigen capable of being recognized by ananalyte (e.g., an antibody analyte, such as a disease-specific antibodyor an auto-antibody) and the second particles comprise a protein orantibody capable of recognizing the analyte. For example, the secondparticle can comprise a protein that is an antibody-binding protein,such as Protein A, Protein G, or Protein L, or an antibody that binds toan antibody constant region (e.g., an anti-IgG or anti-IgM antibody).

In certain embodiments, the population of first particles is in solid(e.g., lyophilized) form. In certain embodiments, the population ofsecond particles is in solid (e.g., lyophilized) form. In certainembodiments, the population of first particles and the population ofsecond particles are in solid (e.g., lyophilized) form.

In certain embodiments, the kit further comprises a container (e.g., atest tube, bottle, or cuvette) that comprises said population of firstparticles, said population of second particles, or both populations ofsaid first and said second particles. In certain embodiments, the kitfurther comprises a rotor, wherein said rotor comprises or is capable ofholding a container (e.g., a cuvette) that comprises said population offirst particles, said population of second particles, or bothpopulations of said first and said second particles.

In certain embodiments, the kit further comprises instructions (e.g.,instructions for using the contents of the kit to carry out a method ofthe invention).

In yet another aspect, the invention provides mixtures comprising apopulation of first particles, a population of second particles, and,optionally, an analyte. In certain embodiments, the mixture is part of asolution-based competition assay. For example, in certain embodiments,the first and second particles are capable of forming multi-particlecomplexes wherein free analyte disrupts (e.g., competitively inhibits)formation of said multi-particle complexes. In other embodiments, themixture is part of a solution-based sandwich assay (e.g., a direct orindirect sandwich assay). For example, in certain embodiments, the firstand second particles are capable of forming multi-particle complexescomprising a first particle, a second particle, and an analyte. Incertain related embodiments, the first and second particles are capableof forming multi-particle complexes, wherein said first particle andsaid second particle each bind to the same analyte, and wherein saidanalyte links said first particle to said second particle.

In certain embodiments, said first and/or second particles aredetectable in suspension. For example, in certain embodiments, the firstand/or second particles scatter light or comprise a detectable color(e.g., visually or spectroscopically detectable color). In certainembodiments, the first and/or second particles comprise a label (e.g., afluorescent label). In certain embodiments, both the first and secondparticles comprise a label (e.g., different fluorescent labels ordifferent chromophores). In certain embodiments, the first particlescomprise a donor chromophore and the second particles comprise anacceptor chromophore, or vice verse, wherein the donor and acceptorchromophores are suitable for detecting interaction between the firstand second particles by means of Förster Resonance Energy Transfer(FRET) analysis.

In certain embodiments, the first and/or second particles are colloidalparticles (e.g., colloidal nanoparticles, nanotubes, core-shellstructure particles, or hollow nanospheres). In certain embodiments, thefirst and/or second particles comprise gold, silver, platinum, copper,or mixed metal. In certain embodiments, the first and/or secondparticles comprise latex, polystyrene, polycarbonate, polyvinylidenefluoride, silica, a polymer having properties similar to any of theforegoing polymers, or a composite thereof. In certain embodiments, thefirst particles comprise gold, silver, platinum, copper, or mixed metal,and the second particles comprise latex, polystyrene, polycarbonate,polyvinylidene fluoride, silica, a polymer having properties similar toany of the foregoing polymers, or a composite thereof.

In certain embodiments, the first particles and the second particleshave different physico-chemical properties. For example, in certainembodiments, the first particles have a greater buoyancy than the secondparticles, or vice versa. In certain embodiments, the first particlesare smaller than the second particles, or vice versa. For example, incertain embodiments, the second particles have a diameter about 5 toabout 50 times larger than the diameter of the first particles.Alternatively, in certain embodiments, the first particles have adiameter about 5 to about 50 times larger than the diameter of thesecond particles. In certain embodiments, the first particles have agreater density than the second particles, or vice versa. In certainembodiments, the first particles are smaller and more buoyant than thesecond particles. In certain embodiments, the first particles aresmaller, more dense, and more buoyant than the second particles.

In certain embodiments, the first particles comprise an analyte and thesecond particles comprise a corresponding analyte-binding agent, such asan antibody, antigen, polypeptide, polynucleotide, nucleoprotein,aptamer, or ligand (e.g., a carbohydrate, lipid, steroid, vitamin, orother small molecule ligand). In other embodiments, both the first andsecond particles comprise an analyte-binding agent, such as an antibody,antigen, polypeptide, polynucleotide, nucleoprotein, aptamer, or ligand(e.g., a carbohydrate, lipid, steroid, vitamin, or other small moleculeligand), wherein the analyte-binding agents bind to the same analyte.

In certain embodiments, the first and second particles comprisedifferent analyte-binding agents. In certain embodiments, the first andsecond particles comprise analyte-binding agents capable of binding todifferent parts of the same analyte (e.g., different domains, differentepitopes, different subunits or molecules in a complex analyte, etc.).In certain embodiments, the first and second particles compriseanalyte-binding agents capable of simultaneously binding to the sameanalyte. In certain embodiments, the first particles comprise a firstantibody capable of recognizing a first epitope on the analyte, thesecond particles comprise a second antibody capable of recognizing asecond epitope on the analyte, and the first and second epitopes aredifferent (e.g., non-overlapping). For example, the first and secondepitopes can be on different surfaces of a simple analyte or differentsubunits or molecules in a complex analyte. In other embodiments, thefirst particles comprise an antigen capable of being recognized by ananalyte (e.g., an antibody analyte, such as a disease-specific antibodyor an auto-antibody) and the second particles comprise a protein orantibody capable of recognizing the analyte. For example, the secondparticle can comprise a protein that is an antibody-binding protein,such as Protein A, Protein G, or Protein L, or an antibody that binds toan antibody constant region (e.g., an anti-IgG or anti-IgM antibody).

The invention and additional embodiments thereof will be set forth ingreater detail in the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict a sandwich assay of the invention. FIG. 1A shows apopulation of first particles and a population of second particles mixedwith an analyte. The first and second particles differ from one anotheron the basis of buoyancy, size, and density. The branched structuresprojecting from the first and second particles are analyte-bindingagents. FIG. 1B shows the formation of complexes between first particlesand second particles, wherein free analyte molecules bind to both firstand second particles and thereby facilitate complex formation. FIG. 1Cshows how the application of force, such as gravity or centrifugalforce, can lead to separation of first particles from second particles,with the larger particles leaving solution to form a pellet along withany complexes that include the larger particles. FIG. 1D shows onerelationship between (1) the absorbance of the mixture of first andsecond particles and analyte, and (2) the concentration of analyte inthe mixture, following the application of an appropriate force.Initially, the absorbance of the mixture goes down as the analyteconcentration goes up, reflecting the fact that increased analyteconcentration leads to increased complex formation, with the complexesbeing pelletted upon application of force.

FIGS. 2A-2E depict a competition assay of the invention. FIG. 2A shows apopulation of first particles and a population of second particles,wherein particles of the first population are coated with analyte andparticles of the second population are coated with correspondinganalyte-binding molecules. The particles of the two populations furtherdiffer from one another on the basis of buoyancy, size, and density.FIG. 2B shows the formation of complexes between particles of the firstand second populations upon mixing. FIG. 2C shows free analyte moleculescompeting with particles of the first population for binding to theanalyte-binding molecules present on particles of the second population,and thereby preventing complex formation and/or disrupting complexesformed between particles of the first and second populations. FIG. 2Dshows how the application of force, such as gravity or centrifugalforce, can lead to separation of first and second particles, with thelarger particles leaving solution to form a pellet along with anycomplexes that include the larger particles. FIG. 2E shows onerelationship between (1) the absorbance of the mixture of first andsecond particles and free analyte, and (2) the concentration of freeanalyte in the mixture, following the application of an appropriateforce. Initially, the absorbance of the mixture goes up as the freeanalyte concentration goes up, reflecting the fact that increasedanalyte concentration leads to decreased complex formation, and thusfewer complexes being pelletted upon application of force.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms shall have the following meanings.

The term “analyte” refers to a substance potentially present in a samplethat can be detected and/or quantified by means of an analyticalprocedure. Analytes that can be detected using the methods of theinvention include, but are not limited to, antigens (e.g.,disease-related antigens), antibodies (e.g., disease-relatedantibodies), macromolecules (e.g., proteins, nucleic acids,carbohydrates, lipids, and combinations thereof), complexes (e.g.,multi-protein complexes, nucleoprotein complexes, complexes comprisingcarbohydrates, lipids, prosthetic groups or other small molecules,etc.), particles (e.g., viral particles or apoptotic bodies), vesicles,cells, and fragments thereof. As used herein, a “complex analyte” is ananalyte that consists of a complex (e.g., a multi-protein complex,nucleoprotein complexes, macromolecular complex, etc.).

The term “antibody” refers to a protein comprising an immunoglobulindomain and an antigen binding site. Thus, the term includes, but is notlimited to, complete antibodies of any isotype (e.g., IgG, IgM, IgA,IgE, IgD), fragments thereof (e.g., Fab, Fab², Fc), single-chainantibodies (e.g., Fv), modified antibodies, and fusion proteinscomprising an immunoglobulin domain and an antigen binding site.

The term “protein” is used interchangeably with the term “polypeptide”and encompasses full-length proteins, protein domains, proteinfragments, mutant proteins, and modified proteins (e.g., proteinscomprising chemically modified amino acids or non-naturally occurringamino acids).

The terms “nucleic acid,” “oligonucleotide,” and “polynucleotide” areused interchangeably and encompass DNA, RNA, and cDNA, whethersingle-stranded or double-stranded, as well as nucleic acids comprisingchemically modified bases or non-naturally occurring bases (e.g., LNA,PNA, etc.).

Additional terms shall be defined, as needed, in the detaileddescription that follows.

Methods

The present invention is based, in part, on the discovery that particleshaving different physical or physico-chemical properties, such asbuoyancy, size, density, spectral characteristics and/or bindingproperties, can be used in solution-based sandwich assays for rapid,qualitative and/or quantitative detection of analytes. The presentinvention is also based, in part, on the discovery that particles havingdifferent physical or physico-chemical properties, such as buoyancy,size, density, spectral characteristics and/or binding properties, canbe used in solution-based competition assays for rapid, qualitativeand/or quantitative detection of analytes.

Accordingly, in one aspect, the present invention provides methods ofdetecting an analyte in a sample. The methods comprise mixing a samplewith a population of first particles and a population of secondparticles to form a suspension. As used herein, the term “suspension”refers to a liquid mixture in which first and second particles are ableto interact with one another and with any analyte that may be present ina sample. As used herein, the term “interact,” as it relates toparticles and analytes, means to collide and, if appropriate, bind(e.g., form non-covalent or covalent chemical bonds) to one another.

Typically, the first particles and/or the second particles arenanoparticles, nanotubes, hollow nanospheres, or core-shell structureparticles. In certain embodiments, the first particles and/or the secondparticles are colloidal nanoparticles, nanotubes, hollow nanospheres, orcore-shell structure particles. As used herein, the terms “colloidalnanoparticles,” “colloidal hollow nanospheres,” and “colloidalcore-shell structure particles” refer to nanoparticles, hollownanospheres, and core-shell structure particles, respectively, that havea diameter of about 1 nm to about 500 nm and remain in suspension inaqueous media. As used herein, the term “colloidal nanotube” refers tonanotubes that have a diameter of about 1 nm to about 500 nm and alength of about 1 nm to about 500 nm and remain in suspension in aqueousmedia. In general, colloidal nanoparticles, nanotubes, hollownanospheres, or core-shell structure particles maintain a homogenousappearance, but do not dissolve, in aqueous media.

In certain embodiments, the first particles comprise gold, silver,platinum, a metal having similar properties, or a composite thereof. Incertain embodiments, the first and second particles comprise gold,silver, platinum, a metal having similar properties, or a compositethereof. For example, in certain embodiments, the first and/or secondparticles are colloidal nanoparticles, nanotubes, hollow nanospheres, orcore-shell structure particles that comprise gold, silver, platinum, ametal having similar properties, or a composite thereof. In certainembodiments, the first particles comprise latex, polystyrene,polycarbonate, polyvinylidene fluoride, silica, a polymer havingproperties similar to any of the foregoing polymers, or a compositethereof. In certain embodiments, the first and second particles compriselatex, polystyrene, polycarbonate, polyvinylidene fluoride, silica, apolymer having properties similar to any of the foregoing polymers, or acomposite thereof. In certain related embodiments, the first and/orsecond particles are colloidal nanoparticles, nanotubes, hollownanospheres, or core-shell structure particles that comprise latex,polystyrene, polycarbonate, polyvinylidene fluoride, silica, a polymerhaving properties similar to any of the foregoing polymers, or acomposite thereof.

In certain embodiments, the first and second particles have differentcompositions. For example, in certain embodiments, the first particlescomprise gold, silver, platinum, a metal having similar properties, or acomposite thereof, and the second particles comprise latex, polystyrene,polycarbonate, polyvinylidene fluoride, silica, a polymer havingproperties similar to any of the foregoing polymers, or a compositethereof.

In certain embodiments, the first and second particles have differentsizes. For example, in certain embodiments, the first particles have anaverage diameter of about 1 nm to about 200 nm and the second particleshave an average diameter of about 200 nm to about 2000 nm. In certainembodiments, the first particles have an average diameter of about 2 nmto about 150 nm, about 3 nm to about 100 nm, about 4 nm to about 70 nm,or about 5 nm to about 40 nm, and the second particles have an averagediameter of about 220 nm to about 1800 nm, about 240 nm to about 1600nm, about 260 nm to about 1400 nm, about 280 nm to about 1200 nm, about300 nm to about 1000 nm, about 320 nm to about 900 nm, about 340 nm toabout 800 nm, about 350 nm to about 700 nm, about 360 nm to about 600nm, about 370 nm to about 500 nm, about 380 nm to about 450 nm, about390 nm to about 425 nm, or about 400 nm.

In certain embodiments, the first particles are colloidal nanoparticles,nanotubes, hollow nanospheres, or core-shell structure particles thatcomprise gold, silver, platinum, a metal having similar properties, or acomposite thereof, and have an average diameter of about 1 nm to about200 nm, about 2 nm to about 150 nm, about 3 nm to about 100 nm, about 4nm to about 70 nm, or about 5 nm to about 40 nm. In certain embodiments,the second particles comprise latex, polystyrene, polycarbonate,polyvinylidene fluoride, silica, a polymer having properties similar toany of the foregoing polymers, or a composite thereof, and have anaverage diameter of about 200 nm to about 2000 nm, about 220 nm to about1800 nm, about 240 nm to about 1600 nm, about 260 nm to about 1400 nm,about 280 nm to about 1200 nm, about 300 nm to about 1000 nm, about 320nm to about 900 nm, about 340 nm to about 800 nm, about 350 nm to about700 nm, about 360 nm to about 600 nm, about 370 nm to about 500 nm,about 380 nm to about 450 nm, about 390 nm to about 425 nm, or about 400nm.

In certain embodiments, the second particles have an average diameterthat is about 2 times larger than the average diameter of the firstparticles. In other embodiments, the second particles have an averagediameter that is about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 60, 70, 80, 90, 100, or more times larger than the averagediameter of the first particles.

In certain embodiments, the first particles are colloidal nanoparticles,nanotubes, hollow nanospheres, or core-shell structure particles thatcomprise gold, silver, platinum, a metal having similar properties, or acomposite thereof, and have an average diameter about 2 to about 100,about 3 to about 80, about 4 to about 65, about 5 to about 50 timessmaller than the average diameter of the second particles. In certainembodiments, the second particles comprise latex, polystyrene,polycarbonate, polyvinylidene fluoride, silica, a polymer havingproperties similar to any of the foregoing polymers, or a compositethereof, and have an average diameter about 2 to about 100, about 3 toabout 80, about 4 to about 65, about 5 to about 50 times larger than theaverage diameter of the first particles.

In certain embodiments, the first and second particles have differentdensities. For example, in certain embodiments, the first particles havea density about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times greaterthan the density of the second particles.

In certain embodiments, the first particles are colloidal nanoparticles,nanotubes, hollow nanospheres, or core-shell structure particles thatcomprise gold, silver, platinum, a metal having similar properties, or acomposite thereof, and have a density about 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, or more times greater than the density of the second particles. Incertain embodiments, the second particles comprise latex, polystyrene,polycarbonate, polyvinylidene fluoride, silica, a polymer havingproperties similar to any of the foregoing polymers, or a compositethereof, and have a density about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore times smaller than the density of the first particles.

In certain embodiments, the first and second particles have differentbuoyancies in aqueous media. For example, in certain embodiments, thefirst particles have an average buoyancy about 1.5 to about 250 timesgreater than the average buoyancy of the second particles. In certainembodiments, the first particles have an average buoyancy about 2 toabout 225, about 3 to about 200, about 4 to about 175, about 5 to about150, about 6 to about 140, about 7 to about 130, about 8 to about 120,about 9 to about 110, about 10 to about 100, about 20 to about 90, about30 to about 80, about 40 to about 70, about 50 to about 60, or about 55times greater than the average buoyancy of the second particles.

In certain embodiments, the first particles are colloidal nanoparticles,nanotubes, hollow nanospheres, or core-shell structure particles thatcomprise gold, silver, platinum, a metal having similar properties, or acomposite thereof, and have an average buoyancy about 1.5 to about 250,about 2 to about 225, about 3 to about 200, about 4 to about 175, about5 to about 150, about 6 to about 140, about 7 to about 130, about 8 toabout 120, about 9 to about 110, about 10 to about 100, about 20 toabout 90, about 30 to about 80, about 40 to about 70, about 50 to about60, or about 55 times greater than the average buoyancy of the secondparticles. In certain embodiments, the second particles comprise latex,polystyrene, polycarbonate, polyvinylidene fluoride, silica, a polymerhaving properties similar to any of the foregoing polymers, or acomposite thereof, and have an average buoyancy about 1.5 to about 250times, about 2 to about 225, about 3 to about 200, about 4 to about 175,about 5 to about 150, about 6 to about 140, about 7 to about 130, about8 to about 120, about 9 to about 110, about 10 to about 100, about 20 toabout 90, about 30 to about 80, about 40 to about 70, about 50 to about60, or about 55 times smaller than the average buoyancy of the firstparticles.

The labels of “first” and “second” particles are used in the foregoingembodiments in a manner that is arbitrary between one embodiment and thenext. Thus, although the first particles are described in the variousembodiments as having a smaller size, greater density, and greaterbuoyancy compared to the second particles, these characteristics are notexclusively linked to first particles. For example, first particles canhave a smaller size, smaller density, and greater buoyancy compared tosecond particles. Alternatively, first particles can have a larger size,smaller density, and greater buoyancy compared to second particles. Inaddition, first particles can have the same size, same density, and/orsame buoyancy compared to second particles. Thus, as persons skilled inthe art will appreciate, the first and second particles can have any mixof relative physico-chemical properties provided that (1) they havedifferent binding properties and (2) complexes formed between first andsecond particles have a buoyancy that is sufficiently low as to allowsedimentation of the complexes without concomitant sedimentation of atleast the first particles, at least the second particles, or eitherfirst or second particles.

In certain embodiments, the first and second particles are capable offorming a multi-particle complex. As used herein, a “multi-particlecomplex” is any molecular aggregate comprising at least one firstparticle and at least one second particle, wherein the first and secondparticles are bound to one another in either a direct or indirectmanner. In certain embodiments, the first and second particles arecapable of forming a multi-particle complex by means of a direct bindinginteraction. The term “direct binding,” as used in this regard, refersto any binding that does not require an additional molecule separatefrom the first and second particles to bridge the interaction. Thus, ifa first particle has a molecule A covalently bound or otherwise stablylinked to its surface and a second particle has a molecule B covalentlybound or otherwise stably linked to its surface, the binding of moleculeA to molecule B constitutes a direct binding interaction between thefirst and second particles.

In other embodiments, the first and second particles are capable offorming a complex by means of an indirect binding interaction. The term“indirect binding,” as used in this regard, refers to any binding thatrequires one or more molecules separate from the first and secondparticles to bridge the interaction. Thus, if a first particle binds toa separate molecule C (e.g., via a molecule A covalently linked to thesurface of the particle) and a second particle also binds to a moleculeC (e.g., via a molecule B covalently linked to the surface of theparticle), the binding of molecule A on a first particle to molecule Bon a second particle via intermediate molecule C constitutes an indirectbinding interaction between the first and second particles.

In certain embodiments, the first particles, second particles, or bothfirst and second particles comprise an analyte-binding agent, such as anantibody, antigen, polypeptide, polynucleotide, nucleoprotein, aptamer,or ligand (e.g., a carbohydrate, lipid, steroid, vitamin, or other smallmolecule ligand). Thus, as suitable for solution-based sandwich assays,the first and second particles can comprise different analyte-bindingagents. For example, in certain embodiments, the first and secondparticles comprise analyte-binding agents capable of binding todifferent parts (e.g., different domains, different epitopes, differentsubunits or molecules in a complex analyte, etc.) of the same analyte.In certain embodiments, the first and second particles compriseanalyte-binding agents capable of simultaneously binding to the sameanalyte molecule.

Thus, in certain embodiments, the first particles comprise a firstantibody capable of recognizing a first epitope on an analyte (e.g., adisease-associated antigen or antibody, a virus or viral antigen, amicroorganism or antigen thereof, etc.), the second particles comprise asecond antibody capable of recognizing a second epitope on the sameanalyte, and the first and second epitopes are different (e.g.,non-overlapping, or overlapping in a minimal way that does not preventthe analyte from simultaneously binding to a first particle and a secondparticle). In other embodiments, the first particles comprise apolypeptide capable of binding to a first surface on an analyte (e.g., adisease-associated antigen or antibody, a virus or viral antigen, amicroorganism or antigen thereof, etc.), the second particles comprise apolypeptide capable of binding to a second surface on the same analyte,and the first and second surfaces are different (e.g., non-overlapping,or overlapping in a minimal way that does not prevent the analyte fromsimultaneously binding to a first particle and a second particle). Instill other embodiments, the first particles comprise a polynucleotidecapable of binding to a first portion of an analyte (e.g., a firstportion of a polynucleotide analyte, such as a disease-associatedpolynucleotide, or a nucleoprotein analyte, such as a viral particle),the second particles comprise a polynucleotide capable of binding to asecond portion of the same analyte, and the first and second portionsare different (e.g., non-overlapping, or overlapping in a minimal waythat does not prevent the analyte from simultaneously binding to a firstparticle and a second particle).

Persons skilled in the art will recognize that the first and secondparticles need not comprise the same type of analyte-binding agent, andthat many different combinations of first particle analyte-binding agentand second particle analyte-binding agent are possible. Accordingly, thefirst particle can comprise an antibody while the second particlecomprises, for example, an antigen, polypeptide, polynucleotide,nucleoprotein, aptamer, or ligand. Similarly, the first particle cancomprise an antigen, while the second particle comprise, for example, anantibody, polypeptide, polynucleotide, nucleoprotein, aptamer, orligand. Alternatively, the first particle can comprise a polynucleotidewhile the second particle comprises, for example, an antibody, antigen,polypeptide, nucleoprotein, aptamer, or ligand. And so on.

Alternatively, as suitable for solution-based competition assays, thefirst particles can comprise an analyte while the second particlescomprise a corresponding analyte-binding agent. The analyte can be anytype of analyte described herein (e.g., antigens (e.g., disease-relatedantigens), antibodies (e.g., disease-related antibodies), macromolecules(e.g., proteins, nucleic acids, carbohydrates, lipids, and combinationsthereof), complexes (e.g., multi-protein complexes, nucleoproteincomplexes, complexes comprising carbohydrates, lipids, prosthetic groupsor other small molecules, etc.), particles (e.g., viral particles orapoptotic bodies), vesicles, cells, and fragments thereof). Similarly,the corresponding analyte-binding agent can take many different forms,such as an antibody, polypeptide, polynucleotide, nucleoprotein,aptamer, or ligand (e.g., a carbohydrate, lipid, steroid, vitamin, orother small molecule ligand).

Thus, in certain embodiments, the first particles comprise an antibody(e.g., a disease-related antibody, such as an autoimmune-antibody) whilethe second particles comprises an antigen recognized by the antibody(e.g., a self antigen). In other embodiments, the first particlescomprise a viral antigen or viral particle, while the second particlescomprise an antibody, polypeptide, or polynucleotide that specificallybinds to the viral antigen and/or viral particle. In still otherembodiments, the first particles comprise a disease-related antigen(e.g., an antigen from an infectious microorganism), while the secondparticles comprise a polypeptide or antibody that binds to thedisease-related antigen.

Again, persons skilled in the art will recognize that there are manydifferent combinations of analyte and analyte-binding agents that can beattached to the first and second particles for use in the methods of theinvention. Accordingly, the present invention is not limited to theforegoing embodiments, but instead is meant to encompass the manydifferent analyte and analyte-binding agent combinations described orsuggested by the present disclosure.

The methods of the invention further comprise removing from thesuspension multi-particle complexes formed after mixing the sample withthe population of first particles and the population of secondparticles. In certain embodiments, the multi-particle complexes areremoved from the suspension by sedimentation. For example, in certainembodiments, the suspension is allowed to rest for a time sufficient toallow gravity to act upon and thereby sediment (i.e., pellet) themulti-particle complexes. In other embodiments, a force is applied tothe suspension to pellet the multi-particle complexes. For example, incertain embodiments, centrifugal force is applied to the suspension,causing any multi-particle complexes present to sediment. In certainembodiments, the suspension is centrifuged (e.g., by spinning thesuspension in a rotor) to achieve sedimentation of the multi-particlecomplexes. In still other embodiments, the multi-particle complexes areremoved from the suspension by magnetic force (e.g., when the first orsecond particles comprise ferromagnetic or paramagnetic materials) or byan electric field (e.g., for charge-based separation of complexes fromone or both types of particles).

In certain embodiments, removing multi-particle complexes from thesuspension comprises removing from the suspension either first particles(e.g., free first particles and first particles present in amulti-particle complex) or second particles (e.g., free second particlesand second particles present in a multi-particle complex). As usedherein, a “free first particle” or a “free second particle” is a firstparticle or a second particle, respectively, that is not part of amulti-particle complex. Since the sedimentation of a particle isdirectly proportional to the square of the particle diameter (assumingconstant particle density), applying a force to the suspensionsufficient to sediment first particles or second particles results insedimentation of larger complexes that comprise one or more of saidfirst or second particles. Thus, the relative buoyancy of firstparticles and second particles can be selected such that, for a givensedimentation force, either the first particles or the second particles(but not both types of particles) sediment along with any multi-particlecomplexes comprising one or more of said first particles and one or moreof said second particles. Accordingly, in certain embodiments, themethods of the invention comprise removing from the suspension (e.g., byapplication of an appropriate sedimentation force) either the firstparticles or the second particles. For example, in certain embodiments,the buoyancy of the first particles is greater than the buoyancy of thesecond particles and the force applied to the suspension (e.g.,centrifugal force) causes sedimentation of second particles (i.e., freesecond particles and any complexes comprising at least one secondparticle), but not sedimentation of free first particles.

In other embodiments, removing multi-particle complexes from thesuspension does not comprise sedimentation of either first particles(i.e., free first particles) or second particles (i.e., free secondparticles). For example, in certain embodiments, the force applied tothe suspension causes sedimentation of multi-particle complexes but isinsufficient to cause sedimentation of either free first particles orfree second particles.

In certain embodiments, free first particles remain in suspension atcentrifugal forces of about 1400 g or less, about 1500 g or less, about1600 g or less, about 1700 g or less, about 1800 g or less, about 1900 gor less, about 2000 g or less, about 2100 g or less, about 2200 g orless, about 2300 g or less, about 2400 g or less, or about 2500 g orless.

In certain embodiments, free second particles remain in suspension atcentrifugal forces of about 1000 g or less, and sediment out ofsuspension at centrifugal forces higher than about 1000 g. In certainembodiments, free second particles remain in suspension at centrifugalforces of about 1100 g or less, and sediment out of suspension atcentrifugal forces higher than about 1100 g. In certain embodiments,free second particles remain in suspension at centrifugal forces ofabout 1200 g or less, and sediment out of suspension at centrifugalforces higher than about 1200 g. In certain embodiments, free secondparticles remain in suspension at centrifugal forces of about 1300 g orless, and sediment out of suspension at centrifugal forces higher thanabout 1300 g. In certain embodiments, free second particles remain insuspension at centrifugal forces of about 1350 g or less, and sedimentout of suspension at centrifugal forces higher than about 1350 g. Incertain embodiments, free second particles remain in suspension atcentrifugal forces of about 1400 g or less, and sediment out ofsuspension at centrifugal forces higher than about 1400 g. In certainembodiments, free second particles remain in suspension at centrifugalforces of about 1450 g or less, and sediment out of suspension atcentrifugal forces higher than about 1450 g. In certain embodiments,free second particles remain in suspension at centrifugal forces ofabout 1500 g or less, and sediment out of suspension at centrifugalforces higher than about 1500 g. In certain embodiments, free secondparticles remain in suspension at centrifugal forces of about 1550 g orless, and sediment out of suspension at centrifugal forces higher thanabout 1550 g.

In certain embodiments, free first particles remain in suspension atcentrifugal forces of about 1000 g or less, while free second particlessediment out of suspension at centrifugal forces of about 500 g to about1000 g. In certain embodiments, free first particles remain insuspension at centrifugal forces of about 1100 g or less, while freesecond particles sediment out of suspension at centrifugal forces ofabout 600 g to about 1100 g. In certain embodiments, free firstparticles remain in suspension at centrifugal forces of about 1200 g orless, while free second particles sediment out of suspension atcentrifugal forces of about 700 g to about 1200 g. In certainembodiments, free first particles remain in suspension at centrifugalforces of about 1300 g or less, while free second particles sediment outof suspension at centrifugal forces of about 800 g to about 1300 g. Incertain embodiments, free first particles remain in suspension atcentrifugal forces of about 1350 g or less, while free second particlessediment out of suspension at centrifugal forces of about 850 g to about1350 g. In certain embodiments, free first particles remain insuspension at centrifugal forces of about 1400 g or less, while freesecond particles sediment out of suspension at centrifugal forces ofabout 900 g to about 1400 g. In certain embodiments, free firstparticles remain in suspension at centrifugal forces of about 1450 g orless, while free second particles sediment out of suspension atcentrifugal forces of about 950 g to about 1450 g. In certainembodiments, free first particles remain in suspension at centrifugalforces of about 1500 g or less, while free second particles sediment outof suspension at centrifugal forces of about 1000 g to about 1500 g. Incertain embodiments, free first particles remain in suspension atcentrifugal forces of about 1550 g or less, while free second particlessediment out of suspension at centrifugal forces of about 1050 g toabout 1550 g. In certain embodiments, free first particles remain insuspension at centrifugal forces of about 1600 g or less, while freesecond particles sediment out of suspension at centrifugal forces ofabout 1100 g to about 1600 g. In certain embodiments, free firstparticles remain in suspension at centrifugal forces of about 1650 g orless, while free second particles sediment out of suspension atcentrifugal forces of about 1150 g to about 1650 g. In certainembodiments, free first particles remain in suspension at centrifugalforces of about 1700 g or less, while free second particles sediment outof suspension at centrifugal forces of about 1200 g to about 1700 g. Incertain embodiments, free first particles remain in suspension atcentrifugal forces of about 1800 g or less, while free second particlessediment out of suspension at centrifugal forces of about 1300 g toabout 1800 g. In certain embodiments, free first particles remain insuspension at centrifugal forces of about 1900 g or less, while freesecond particles sediment out of suspension at centrifugal forces ofabout 1400 g to about 1900 g. In certain embodiments, free firstparticles remain in suspension at centrifugal forces of about 2000 g orless, while free second particles sediment out of suspension atcentrifugal forces of about 1500 g to about 2000 g.

In certain embodiments, removing multi-particle complexes from thesuspension is achieved by applying a centrifugal force of about 1000 gto about 2500 g to the suspension. In certain embodiments, removingmulti-particle complexes from suspension is achieved by applying acentrifugal force of about 1200 g to about 2000 g, about 1350 g to about2150 g, about 1500 g to about 2300 g, about 1000 g to about 1200 g,about 1100 g to about 1300 g, about 1200 g to about 1400 g, about 1300 gto about 1500 g, about 1400 g to about 1600 g, about 1500 g to about1700 g, about 1600 g to about 1800 g, about 1700 g to about 1900 g,about 1800 g to about 2000 g, about 1900 g to about 2100 g, about 2000 gto about 2200 g, about 2100 g to about 2300 g, about 2200 g to about2400 g, or about 2300 g to about 2500 g to the suspension.

In certain embodiments, removing free first particles or free secondparticles from the suspension is achieved by applying a centrifugalforce of about 1000 g to about 2500 g. In certain embodiments, removingfirst particles or second particles from suspension is achieved byapplying a centrifugal force of about 1200 g to about 2000 g, about 1350g to about 2150 g, about 1500 g to about 2300 g, about 1000 g to about1200 g, about 1100 g to about 1300 g, about 1200 g to about 1400 g,about 1300 g to about 1500 g, about 1400 g to about 1600 g, about 1500 gto about 1700 g, about 1600 g to about 1800 g, about 1700 g to about1900 g, about 1800 g to about 2000 g, about 1900 g to about 2100 g,about 2000 g to about 2200 g, about 2100 g to about 2300 g, about 2200 gto about 2400 g, or about 2300 g to about 2500 g to the suspension.

In certain embodiments, applying a centrifugal force of about 1000 g toabout 2500 g (e.g., about 1300 g to about 1800 g) to the suspensionresults in sedimentation of first particles (e.g., free first particlesand first particles present in a complex) or second particles (e.g.,free second particles and second particles present in a complex), butnot both. In certain embodiments, applying a centrifugal force of about1000 g to about 2500 g (e.g., about 1300 g to about 1800 g) to thesuspension does not result in sedimentation of either first particles(i.e., free first particles) or second particles (i.e., free secondparticles).

The methods of the invention further comprise detecting the presence offirst and/or second particles remaining in suspension. For example, incertain embodiments, the first and/or second particles scatter light. Insuch embodiments, the presence of first and/or second particles insuspension can be detected, e.g., by passing light through thesuspension and measuring the amount of light scattering as compared toan equivalent suspension that lacks first and second particles. In otherembodiments, the presence of first and/or second particles in suspensioncan be detected, e.g., by passing light through the suspension andmeasuring absorbance (e.g., absorbance at a particular wavelength oracross a range of wavelengths). In other embodiments, the first and/orsecond particles comprise a label, such as a fluorescent label. In suchembodiments, the presence of first and/or second particles can bedetected, e.g., by exciting the fluorescent label and detecting theresulting fluorescence. In related embodiments, the first and secondparticles can comprise different labels, e.g., fluorescent labels, suchas Qdots, having different emission wavelengths, thereby allowingseparate detection of the first and second particles. In still otherembodiments, the first or second particles comprise a metal (e.g., gold,silver, platinum, a metal having similar properties, or a compositethereof) and are detected using surface-enhanced raman scattering(SERS).

In certain embodiments, detecting the presence of first and/or secondparticles provides a qualitative assessment. In other embodiments,detecting the presence of first and/or second particles provides aquantitative measurement of the amount of first and/or second particlespresent. For example, in certain embodiments, measurements of, e.g.,light scattering, light absorption, fluorescence/luminescence emission,or SERS, allows for the amount of first and/or second particlesremaining in suspension to be determined quantitatively.

In certain embodiments, a decrease in amount of first and/or secondparticles in suspension is indicative of the presence of the analyte inthe sample. For example, in certain embodiments, the assay is a sandwichassay (e.g., a direct or indirect sandwich assay) in which the first andsecond particles form a complex by binding to the same analyte and adecrease in the amount of first and/or second particles in suspension isindicative of the presence of the analyte in the sample. In otherembodiments, an increase in the amount of first and/or second particlesin suspension is indicative of the presence of the analyte in thesample. For example, in certain embodiments, the assay is a competitionassay in which the first particles comprise an analyte, the secondparticles comprise a corresponding analyte-binding agent, and anincrease in the amount of first and/or second particles in suspension isindicative of the presence of the analyte in the sample. As personsskilled in the art will readily understand, the decrease or increase isrelative to an appropriate standard. For example, an appropriatestandard will comprise an equivalent amount of first and secondparticles and an appropriate saline solution or a sample known not tocontain the analyte of interest.

In certain embodiments, the analyte is biological analyte. For example,in certain embodiments, the analyte is a pathogenic antigen or anantibody thereto. Suitable pathogenic antigens can originate fromviruses (e.g., feline leukemia virus, canine parvovirus, foot and mouthvirus, influenza virus, hepatitis a, b, or c virus, HIV virus, humanpapilloma virus, epstein barr virus, rabies virus, etc.), bacteria(e.g., Ehrlichia, Borellia, Anthrax, Salmonella, Bacillus, etc.), fungi,or parasites (e.g., canine heartworm, Giardia lamblia, Plasmodiumfalciparum, African trypanosomiasis, Trypanosoma brucei, etc.). Incertain embodiments, the analyte is a disease-related antigen or anantibody thereto. Disease-related antigens include, but are not limitedto, cancer-related antigens (e.g., PSA, AFP, CA125, CA15-3, CA19-9, CEA,NY-ESO-1, MUC1, GM3, GD2, ERBB2, etc.), cardiovascular disease-relatedantigens (e.g., cardiac troponin, C-reactive protein, CK-MB, fatty acidbinding protein, etc.), or auto-immune disease-related antigens (e.g.,auto-antibodies). In certain embodiments, the analyte is a inflammatoryantigen (e.g., C-reactive protein, MRP14, MRP8, 25F9, etc.). In certainembodiments, the analyte is a pregnancy-related antigen (e.g., a fetalantigen).

In other embodiments, the analyte is a non-biological analyte, such asan environmental analyte (e.g., an environmental contaminant).

In certain embodiments, the analyte is present in a biological sample.Biological samples include, but are not limited to, biological fluids(e.g., blood, serum, urine, cerebrospinal fluid, saliva, etc.), tissuehomogenates, cell lysates, or extracts thereof. In certain embodiments,the analyte is present in an environmental sample, such as a sample ofground water, river, lake, waste water, etc.

In certain embodiments, the methods of the invention are performed in acontainer, such as a tube or a cuvette. In certain embodiments, themethods of the invention are performed using a rotor (e.g., a rotor fora centrifuge). In certain embodiments, the container (e.g., tube orcuvette) fits into a rotor. In other embodiments, the container (e.g.,tube or cuvette) is built into a rotor. In certain related embodiments,the methods of the invention comprise adding the sample to a rotor thatcontains the population of first particles and the population of secondparticles, wherein said mixing occurs in said rotor.

In certain embodiments, the population of first particles is in dry formprior to being mixed with sample. In certain embodiments, the populationof second particles is in dry form prior to being mixed with sample. Incertain embodiments, both the population of first particles and thepopulation of second particles are in dry form prior to being mixed withsample. In certain embodiments, the dry form is a lyophilized bead ofparticles. The size of the bead will depend upon the number and size ofthe particles in the bead. In certain embodiments, a bead comprisesabout 10⁸ to about 10¹², about 10⁹ to about 10¹¹, or about 2×10⁹, about3×10⁹, about 4×10⁹, about 5×10⁹, about 6×10⁹, about 7×10⁹, about 8×10⁹,about 9×10⁹, about 1×10¹⁰, about 2×10¹⁰, about 3×10¹⁰, about 4×10¹⁰,about 5×10¹⁰, about 6×10¹⁰, about 7×10¹⁰, about 8×10¹⁰, about 9×10¹⁰particles, wherein the particles have an average diameter of about 10 toabout 40 nm. In certain embodiments, a bead comprises about 10⁵ to about10⁹, about 10⁶ to about 10⁸, or about 2×10⁶, about 3×10⁶, about 4×10⁶,about 5×10⁶, about 6×10⁶, about 7×10⁶, about 8×10⁶, about 9×10⁶, about1×10⁷, about 2×10⁷, about 3×10⁷, about 4×10⁷, about 5×10⁷, about 6×10⁷,about 7×10⁷, about 8×10⁷, about 9×10⁷ particles, wherein the particleshave an average diameter of about 200 to about 500 nm.

In certain embodiments, the population of first particles and thepopulation of second particles are provided in dry form (e.g., aslyophilized beads) in a rotor, wherein the addition of a liquid sampleto the rotor results in mixing of the sample with the populations offirst and second particles. In certain related embodiments, thepopulation of first particles and the population of second particles areprovided separately, e.g., as separate lyophilized beads located withina mixing chamber of a rotor.

Kits

In another aspect, the invention provides kits. In certain embodiments,the kits comprise a population of first particles and a population ofsecond particles, wherein said populations are suitable for use in themethods of the invention. The first and second particles can be anyfirst and second particles described or suggested herein. Accordingly,in certain embodiments, the first and second particles are capable offorming complexes. In certain embodiments, the first and secondparticles are suitable for use in solution-based competition assays. Forexample, in certain embodiments, the first and second particles arecapable of forming multi-particle complexes wherein free analytedisrupts (e.g., competitively inhibits) formation of said multi-particlecomplexes. In other embodiments, the first and second particles aresuitable for use in solution-based sandwich assays. For example, incertain embodiments, the first and second particles are capable offorming multi-particle complexes comprising a first particle, a secondparticle, and an analyte. In certain related embodiments, the first andsecond particles are capable of forming multi-particle complexes,wherein said first particle and said second particle each bind to thesame analyte, and wherein said analyte links said first particle to saidsecond particle.

In certain embodiments, the first and/or second particles are detectablein suspension. For example, in certain embodiments, the first and/orsecond particles scatter light or comprise a detectable color (e.g.,visually or spectroscopically detectable color). In certain embodiments,the first and/or second particles comprise a label (e.g., fluorescentlabel). In certain embodiments, both the first and second particlescomprise a label (e.g., different fluorescent labels or differentchromophores). In certain embodiments, the first particles comprise adonor chromophore and the second particles comprise an acceptorchromophore, or vice verse, wherein the donor and acceptor chromophoresare suitable for detecting interaction between the first and secondparticles by means of Förster Resonance Energy Transfer (FRET) analysis.

In certain embodiments, the first and/or second particles comprise gold,silver, platinum, a metal having similar properties, or a compositethereof. For example, in certain embodiments, the first and/or secondparticles are colloidal nanoparticles, nanotubes, hollow nanospheres, orcore-shell structure particles that comprise gold, silver, platinum, ametal having similar properties, or a composite thereof. In otherembodiments, the first and/or second particles comprise latex,polystyrene, polycarbonate, polyvinylidene fluoride, silica, a polymerhaving properties similar to any of the foregoing polymers, or acomposite thereof. In certain related embodiments, the first and/orsecond particles are colloidal nanoparticles, nanotubes, hollownanospheres, or core-shell structure particles that comprise latex,polystyrene, polycarbonate, polyvinylidene fluoride, silica, a polymerhaving properties similar to any of the foregoing polymers, or acomposite thereof.

In certain embodiments, the first particles and the second particleshave different physico-chemical properties. For example, in certainembodiments, the first particles have a greater buoyancy than the secondparticles, or vice versa. In certain embodiments, the first particlesare smaller than the second particles, or vice versa. For example, incertain embodiments, the second particles have a diameter about 5 toabout 50 times larger than the diameter of the first particles. In otherembodiments, the first particles have a diameter about 5 to about 50times larger than the diameter of the second particles. In certainembodiments, the first particles have a greater density than the secondparticles, or vice versa. In certain embodiments, the first particlesare smaller and more buoyant than the second particles. In certainembodiments, the first particles are smaller, more dense, and morebuoyant than the second particles.

In certain embodiments, the first particles comprise an analyte and thesecond particles comprise a corresponding analyte-binding agent, such asan antibody, antigen, polypeptide, polynucleotide, nucleoprotein,aptamer, or ligand (e.g., a carbohydrate, lipid, steroid, vitamin, orother small molecule ligand). In other embodiments, both the first andsecond particles comprise an analyte-binding agent, such as an antibody,antigen, polypeptide, polynucleotide, nucleoprotein, aptamer, or ligand(e.g., a carbohydrate, lipid, steroid, vitamin, or other small moleculeligand), wherein the analyte-binding agents bind to the same analyte.

In certain embodiments, the first and second particles comprisedifferent analyte-binding agents. In certain embodiments, the first andsecond particles comprise analyte-binding agents capable of binding todifferent parts of the same analyte (e.g., different domains, differentepitopes, different subunits or molecules in a complex analyte, etc.).In certain embodiments, the first and second particles compriseanalyte-binding agents capable of simultaneously binding to the sameanalyte. For example, in certain embodiments, the first particlescomprise a first antibody capable of recognizing a first epitope on theanalyte, the second particles comprise a second antibody capable ofrecognizing a second epitope on the analyte, and the first and secondepitopes are different (e.g., non-overlapping). For example, the firstand second epitopes can be on different surfaces of a simple analyte ordifferent subunits or molecules in a complex analyte. In otherembodiments, the first particles comprise an antigen capable of beingrecognized by an analyte (e.g., an antibody analyte, such as adisease-specific antibody or an auto-antibody) and the second particlescomprise a protein or antibody capable of recognizing the analyte. Forexample, the second particle can comprise a protein that is anantibody-binding protein, such as Protein A, Protein G, or Protein L, oran antibody that binds to an antibody constant region (e.g., an anti-IgGor anti-IgM antibody).

In certain embodiments, the population of first particles is in solid(e.g., lyophilized) form. In certain embodiments, the population ofsecond particles is in solid (e.g., lyophilized) form. In certainembodiments, the population of first particles and the population ofsecond particles are in solid (e.g., lyophilized) form.

In certain embodiments, the kit further comprises a container (e.g., acuvette) that comprises said population of first particles, saidpopulation of second particles, or both populations of said first andsaid second particles. In certain embodiments, the kit further comprisesa rotor, wherein said rotor comprises a container (e.g., a cuvette) thatcomprises said population of first particles, said population of secondparticles, or both populations of said first and said second particles.

In certain embodiments, the kit further comprises instructions (e.g.,instructions for using the contents of the kit to carry out a method ofthe invention).

Mixtures

In yet another aspect, the invention provides mixtures. In certainembodiments, the mixtures comprise a population of first particles, apopulation of second particles, and, optionally, an analyte. The firstand second particles can be any first and second particles described orsuggested herein. Similarly, the analyte can be any analyte described orsuggested herein. Accordingly, in certain embodiments, the mixture ispart of a solution-based competition assay. For example, in certainembodiments, the first and second particles are capable of formingmulti-particle complexes wherein free analyte disrupts (e.g.,competitively inhibits) formation of said multi-particle complexes. Inother embodiments, mixture is part of a solution-based sandwich assay.For example, in certain embodiments, the first and second particles arecapable of forming multi-particle complexes comprising a first particle,a second particle, and an analyte. In certain related embodiments, thefirst and second particles are capable of forming multi-particlecomplexes, wherein said first particle and said second particle eachbind to the same analyte, and wherein said analyte links said firstparticle to said second particle.

In certain embodiments, the first and/or second particles are detectablein suspension. For example, in certain embodiments, the first and/orsecond particles scatter light or comprise a detectable color (e.g.,visually or spectroscopically detectable color). In certain embodiments,the first and/or second particles comprise a label (e.g., fluorescentlabel). In certain embodiments, both the first and second particlescomprise a label (e.g., different fluorescent labels or differentchromophores). In certain embodiments, the first particles comprise adonor chromophore and the second particles comprise an acceptorchromophore, or vice verse, wherein the donor and acceptor chromophoresare suitable for detecting interaction between the first and secondparticles by means of Förster Resonance Energy Transfer (FRET) analysis.

In certain embodiments, the first and/or second particles comprise gold,silver, platinum, a metal having similar properties, or a compositethereof. For example, in certain embodiments, the first and/or secondparticles are colloidal nanoparticles, nanotubes, hollow nanospheres, orcore-shell structure particles that comprise gold, silver, platinum, ametal having similar properties, or a composite thereof. In otherembodiments, the first and/or second particles comprise latex,polystyrene, polycarbonate, polyvinylidene fluoride, silica, a polymerhaving properties similar to any of the foregoing polymers, or acomposite thereof. In certain related embodiments, the first and/orsecond particles are colloidal nanoparticles, nanotubes, hollownanospheres, or core-shell structure particles that comprise latex,polystyrene, polycarbonate, polyvinylidene fluoride, silica, a polymerhaving properties similar to any of the foregoing polymers, or acomposite thereof.

In certain embodiments, the first particles and the second particleshave different physico-chemical properties. For example, in certainembodiments, the first particles have a greater buoyancy than the secondparticles, or vice versa. In certain embodiments, the first particlesare smaller than the second particles, or vice versa. For example, incertain embodiments, the second particles have a diameter about 5 toabout 50 times larger than the diameter of the first particles. In otherembodiments, the first particles have a diameter about 5 to about 50times larger than the diameter of the second particles. In certainembodiments, the first particles have a greater density than the secondparticles, or vice versa. In certain embodiments, the first particlesare smaller and more buoyant than the second particles. In certainembodiments, the first particles are smaller, more dense, and morebuoyant than the second particles.

In certain embodiments, the first particles comprise an analyte and thesecond particles comprise a corresponding analyte-binding agent, such asan antibody, polypeptide, polynucleotide, nucleoprotein, or ligand(e.g., a carbohydrate, lipid, steroid, vitamin, or other small moleculeligand). In other embodiments, both the first and second particlescomprise an analyte-binding agent, such as an antibody, polypeptide,polynucleotide, nucleoprotein, or ligand (e.g., a carbohydrate, lipid,steroid, vitamin, or other small molecule ligand), wherein theanalyte-binding agents bind to the same analyte.

In certain embodiments, the first and second particles comprisedifferent analyte-binding agents. In certain embodiments, the first andsecond particles comprise analyte-binding agents capable of binding todifferent parts of the same analyte (e.g., different domains, differentepitopes, etc.). In certain embodiments, the first and second particlescomprise analyte-binding agents capable of simultaneously binding to thesame analyte. For example, in certain embodiments, the first particlescomprise a first antibody capable of recognizing a first epitope on theanalyte, the second particles comprise a second antibody capable ofrecognizing a second epitope on the analyte, and the first and secondepitopes are different (e.g., non-overlapping). For example, the firstand second epitopes can be on different surfaces of a simple analyte ordifferent subunits or molecules in a complex analyte. In otherembodiments, the first particles comprise an antigen capable of beingrecognized by an analyte (e.g., an antibody analyte, such as adisease-specific antibody or an auto-antibody) and the second particlescomprise a protein or antibody capable of recognizing the analyte. Forexample, the second particle can comprise a protein that is anantibody-binding protein, such as Protein A, Protein G, or Protein L, oran antibody that binds to an antibody constant region (e.g., an anti-IgGor anti-IgM antibody).

The present invention has been illustrated and described in detail withreference to particular embodiments by way of example only, and not byway of limitation. Those of skill in the art will appreciate thatvarious modifications to the disclosed embodiments are within the scopeand contemplation of the invention of the present disclosure. Therefore,it is intended that the invention be considered as limited only by thescope of the appended claims.

What is claimed is:
 1. A method for detecting the presence of an analytein a sample, wherein the analyte in the sample is free analyte,comprising: mixing the sample with a population of first particles and apopulation of second particles to form a suspension, wherein the firstparticles comprise the analyte in bound form; and wherein the firstparticles and second particles are capable of forming multi-particlecomplexes, removing multi-particle complexes from the suspension, anddetecting the presence of free first particles in the suspension afterremoval of the multi-particle complexes; wherein an increase in theamount of free first particles in the suspension relative to the amountof free first particles present in a negative control suspension isindicative of the presence of the analyte in the sample.
 2. The methodof claim 1, wherein the negative control suspension is prepared by amethod comprising mixing a control sample that does not comprise thefree analyte with the population of first particles and the populationof second particles, and removing multi-particle complexes to form thenegative control suspension.
 3. The method of claim 1, wherein the firstparticles comprise the analyte attached to or coating structuresselected from the group consisting of colloidal nanoparticles,nanotubes, hollow nanospheres, and core-shell structures, wherein thestructures comprise gold, silver, platinum, copper, or a composite ofany of the foregoing metals.
 4. The method of claim 1, wherein thesecond particles comprise latex, polystyrene, polycarbonate,polyacrylate, PVDF, or silica.
 5. The method of claim 1, wherein thesecond particles comprise an antibody, antigen, polypeptide,polynucleotide, nucleoprotein, or aptamer.
 6. The method of claim 5,wherein the second particles comprise an antibody that recognizes anepitope on the analyte.
 7. The method of claim 1, wherein the free firstparticles remain in suspension at centrifugal forces of about 1600 g orless.
 8. The method of claim 1, wherein the first particles are smallerthan the second particles.
 9. The method of claim 1, wherein the secondparticles sediment out of the suspension at centrifugal forces of about1000 g to about 1600 g.
 10. The method of claim 1, wherein the firstparticles have a diameter of about 5 nm to about 40 nm and wherein thesecond particles have a diameter of about 400 nm to about 2000 nm. 11.The method of claim 1, wherein the sample is in a liquid form andwherein the population of the first particles and population of secondparticles are in solid form prior to the mixing.
 12. The method of claim1, wherein the analyte is an antigen selected from canine heartworm,feline leukemia virus, canine parvovirus, C-reactive protein, Giardialamblia, Ehrlichia antigen or antibody, Borrelia antigen or antibody,and cardiac marker antigens.
 13. The method of claim 1, wherein removingthe complex comprises using centrifugal force.
 14. The method of claim1, wherein the population of first particles and population of secondparticles are separate prior to mixing with the sample.
 15. The methodof claim 1, wherein the ratio of the average diameter of secondparticles to the average diameter of first particles is about 5:1 toabout 50:1.